NMR Studies of Proteins

The complete assignment of individual resonances for a protein can, in principle, be achieved by using multidimensional NMR spectroscopic techniques. For simplicity, the following three 2D NMR H techniques will be discussed. They are COSY (Section 13.3), NOESY (Section 13.4), and TOCSY. These techniques allow for the identification of resonances for nuclei that are connected through bonds, those that are in close proximity in space, and those that are within a given spin system, respectively. 

With the backbone assigned, it is necessary to continue with the assignment of the side-chain protons. Commonly, another 2D spectroscopic technique, TOCSY (total correlation spectroscopy), is employed in addition to COSY. This 

In addition to the use of NOESY spectra for the assignment of individual resonances, they can also be used for the determination of the three-dimensional structure of the protein in solution. Up to now we have only dealt with the connectivities along the backbone between protons that are close to each other because of chemical bonding. There are also correlations between amino acids that are far apart in the primary structure but are in close proximity because of the folding found in the tertiary structure. For example, a portion of the chain near the amino terminus can be near the carboxy termi- 

These data can be interpreted as follows. Since the chemical shifts of both resonances from the protein and the inhibitor are concentration dependent, the exchange rate between the free and bound states is fast on the NMR time scale. The observed chemical shifts are a weighted average of the two states [Eq. (10.13)]. For the protein resonances, the fraction bound increases with increasing inhibitor concentration. At zero concentration of the inhibitor, the observed chemical shift is that of the unbound state, while at concentrations above -0.06 M inhibitor, the observed chemical shift is that of the bound state. For the inhibitor, the highest fraction bound occurs at the lower concentrations of the inhibitor and the lowest fraction bound at the higher concentrations. There- 

NMR techniques can thus be used to ascertain the nature of the binding of small molecules to large proteins. Although initial studies involved the simplest of systems, similar analy- 

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Torchia, D. A., 1984. Solid state NMR studies of protein internal dynamics. Annual Review of Biophysics and Bioengineering 13 125—144. 

This kind of shift mapping has been used extensively in NMR studies of protein-ligand interactions. Shuker et al.1/2 used this method to advantage in a lead generation approach that effectively covalently links two... 

Finally, it is worth keeping in mind that the protein may be perturbed reversibly by the conditions of the NMR experiment. For example, a recent study demonstrated that removal of the bulk buffer that typically separates from the sample during magic angle spinning reversibly altered the conformation of the selectivity filter of the KcsA potassium channel addition of buffer to the rotor restored the conformation.101 Based on this observation of a hydration-induced shift in conformation, it seems prudent to maintain high hydration levels for NMR studies of proteins in general. 

Chemical synthesis has provided an additional route to peptides containing halogenat-ed amino acids. Early 19F-NMR studies of proteins were performed on semi-synthetic polypeptides prepared by attachment of fluorinated probes to the polypeptide. For example, Heustis and Raftery modified ribonuclease by trifluoroacetylation of Lys residues 1 and 7. They then used 19F-NMR to study conformational changes brought about by the presence of inhibitors200. In his review, Gerig provides several other examples of this strategy187. 

Siminovitch, D.I. (1998) Solid-state NMR studies of proteins the view from static 2H NMR experiments, Biochemistry and Cell Biology 76, 411-422. 

Because of the ease with which molecular mechanics calculations may be obtained, there was early recognition that inclusion of solvation effects, particularly for biological molecules associated with water, was essential to describe experimentally observed structures and phenomena [32]. The solvent, usually an aqueous phase, has a fundamental influence on the structure, thermodynamics, and dynamics of proteins at both a global and local level [3/]. Inclusion of solvent effects in a simulation of bovine pancreatic trypsin inhibitor produced a time-averaged structure much more like that observed in high-resolution X-ray studies with smaller atomic amplitudes of vibration and a fewer number of incorrect hydrogen bonds [33], High-resolution proton NMR studies of protein hydration in aqueous... 

The NMR studies of proteins lead to information about their primary, secondary, and tertiary structure as well as information about their interactions with substrates. 

VI. NMR Studies of Proteins Containing Polynuclear Copper Centers. 434... 

An interesting recent development is the use of arginine-glutamate salt (typically 50 mmol l-1 L-arginine + 50 mmol l-1 L-glutamate) for NMR studies of proteins using CC probes. This zwitterionic salt not only has much lower conductivity than NaCl but has also been shown to help solubilize proteins that are prone to aggregation.9,10... 

R109 N. Jamin and F. Toma, NMR Studis of Protein-DNA Interactions , p. 83... 

R576 A. J. Wand, J. L. Urbauer, M. R. Ehrhardt and A. L. Lee, NMR Studies of Protein-Peptide Complexes Examples from the Calmodulin System , Drugs Pharm. Sci., 2000,101, 727... 

In liquid-state NMR, spin relaxation due to cross-correlation of two anisotropic spin interactions can provide useful information about molecular structure and dynamics. These effects are manifest as differential line widths or line intensities in the NMR spectra. Recently, new experiments were developed for the accurate measurement of numerous cross-correlated relaxation rates in scalar coupled multi-spin systems. The recently introduced concept of transverse relaxation optimized spectroscopy (TROSY) is also based on cross-correlated relaxation. Brutscher outlined the basic concepts and experimental techniques necessary for understanding and exploiting cross-correlated relaxation effects in macromolecules. In addition, he presented some examples showing the potential of cross-correlated relaxation for high-resolution NMR studies of proteins and nucleic acids. 

Can H cramps NMR become an effective means for conformational analysis of polypeptides and proteins in the solid state In order to answer this question, in this section, we focus on and discuss a study on correlation between the H chemical shifts and the main-chain conformation (secondary structure) of synthetic polypeptides.Homopolypeptides and copolypeptides are useful as models for structural analysis of natural proteins. Homopolypeptide is the simplest and the most basic model polypeptide for NMR studies of proteins. 

Feeney, J. and Birdsall, B. (1993). NMR studies of protein-ligand interactions, in NMR of Macromolecules A Practical Approach (Roberts, G. C. K., Ed.). Oxford University Press, Oxford. 

F Separovic, YH Lam, X Ke, H-K Chan. A solid-state NMR study of protein hydration and stability. Pharm Res 15 1816-1821, 1998. 

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